JP2013139453A - Anti-ngf antibody for treatment of various disorders - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for using an anti-NGF antibody in a treatment of various nerve growth factor (NGF)-related disorders including asthma, arthritis and psoriasis without causing significant adverse effect on an immune system of a patient.SOLUTION: In vivo administration of a therapeutically active amount of an anti-NGF monoclonal antibody (antibody 911), no adverse action has been found on an immune system of an allergy experimental mouse model. Based on the finding, NGF-related diseases including human asthma is treated by a method using an antibody relating to the monoclonal antibody.

Description

(Background of the Invention)
(Field of Invention)
The present invention relates generally to methods of using anti-NGF antibodies for the treatment of various NGF-related diseases including asthma, arthritis, and psoriasis. This method is effective in treating these diseases without causing significant side effects on the patient's immune system.

Related Art Description Nerve Growth Factor (NGF)
Nerve growth factor (NGF) is the first neurotrophin identified and its role in both peripheral and central nerve development and survival is well characterized. NGF has been shown to be an important survival and maintenance factor in the development of peripheral sympathetic and fetal sensory neurons and forebrain basal cholinergic neurons (Smeyne et al., Nature 368: 246-249 (1994) Crowley et al., Cell 76: 1001-1011 (1994)). NGF upregulates neuropeptide expression in sensory neurons (Lindsay and Harmer, Nature 337: 362-364 (1989)), whose activity is mediated through two different membrane-bound receptors. The TrkA tyrosine kinase receptor mediates high affinity binding, and the p75 receptor structurally related to other members of the tumor necrosis factor receptor family mediates low affinity binding (Chao et al., Science 232: 518- 521 (1986)).

In addition to its effects on the nervous system, NGF has become increasingly associated with processes outside the nervous system. For example, NGF increases vascular permeability (Otten et al., Eur. J. Pharmacol. 106: 199-201 (1984)) and enhances T- and B-cell immune responses (Otten et al., Proc. Natl. Acad). Sci. USA 86: 10059-10063 (1989)), induces lymphocyte differentiation and mast cell proliferation to cause release of soluble biological signals from mast cells (Matsuda et al., Proc. Natl. Acad. Sci. USA 85: 6508-6512 (1988); Pearce et al., J. Physiol. 372: 379-393 (1986); Bischoff et al., Blood 79: 2662-2669 (1992); Horigome et al., J. Biol. Chem. 268: 14881-14887 (1993)).
NGF is found in mast cells (Leon et al., Proc. Natl. Acad. Sci. USA 91: 3739-3743 (1994)), B-lymphocytes (Torcia et al., Cell 85: 345-356 (1996)), keratinocytes (Di Marco et al., J. Biol. Chem. 268: 22838-22846) and smooth muscle cells (Ueyama et al., J. Hypertens. 11: 1061-1065 (1993)). NGF receptors have been found in various cell types outside the nervous system. For example, TrkA has been found on human monocytes, T- and B-lymphocytes and mast cells.

  Consistent with the non-neuronal role of NGF, an association between increased NGF levels and various inflammatory symptoms has been observed in human patients along with several animal models. These include systemic lupus erythematosus (Bracci-Laudiero et al., Neuroreport 4: 563-565 (1993)), multiple sclerosis (Bracci-Laudiero et al., Neurosci. Lett. 147: 9-12 (1992)), psoriasis ( Raychaudhuri et al., Acta Derm. Venereol. 78: 84-86 (1998)), arthritis (Falcini et al., Ann. Rheum. Dis. 55: 745-748 (1996)) and asthma (Braun et al., Eur. J. Immunol. 28: 3240-3251 (1998)). Chronic inflammatory symptoms such as these are serious public health problems. For example, in the United States alone, it is estimated that 379.9 million people suffer from arthritis. Current therapies for treating these symptoms are very limited. Understanding the role NGF plays in these diseases will provide new ways to treat them.

  An association between stress and psoriasis has been found. Based on this relationship and the symmetry of skin disorders with disease, an association with the nervous system has been proposed (Raychaudhuri et al., Acta Derm. Venercol. 78: 84-86 (1998)). In particular, it has been suggested that neuropeptides are responsible for the pathogenicity of psoriasis. Researchers have reported an increase in the number of terminal cortical nerves in synchrony with the upregulation of one or more neuropeptides such as substance P (SP), vasoactive intestinal polypeptide (VIP) and CGRP. NGF is also responsible for controlling cutaneous neurosensory and is also known to up-regulate neuropeptides, which means that NGF levels are increased in neuropeptide upregulation and increased skin-reactive nerves seen in psoriasis. This suggests that it is a cause. Indeed, increased expression of NGF has been observed in psoriatic keratinocytes (Raychaudhuri et al., Acta Derm. Venernol. 78: 84-86 (1998)). While NGF normally functions as a keratinocyte survival factor, overexpression of NGF has been suggested to prevent normal cell death and lead to psoriasis (Pincelli et al., J. Derm. Sci. 22: 71 -79 (2000)).

Neuroactive peptides such as substance P (SP) and bioactive compounds released from mast cells such as histamine play a role in both arthritis that occurs naturally in humans and experimentally induced arthritis in animal models. Has been shown by many studies (Levine, J. Science 226: 547-549 (1984)). NGF has been shown to act on degranulation of mast cells (Bruni et al., FEBS Lett. 138: 190-193 (1982)) and release of substance P (Donnerer et al., Neurosci. 49: 693-698 (1982)). This indicates that NGF is the etiology of arthritis.
Consistently, elevated levels of end tissue NGF are associated with both hyperalgesia and inflammation, and are found in many types of arthritis. While it has been reported that NGF is not detected in non-inflammatory synovium, the synovium of patients with rheumatoid arthritis expresses high levels of NGF (Aloe et al., Arch. Rheum. 35: 351-355 ( 1992)). Similar results were seen in rats with experimentally induced rheumatoid arthritis (Aloe et al., Clin. Exp. Rheumatol. 10: 203-204 (1992)). Elevated levels of NGF have been reported in transgenic arthritic mice with an increased number of mast cells (Aloe et al., Int. J. Tissue Reaction-Exp. Clin. Aspects 15: 139-143 (1993)). However, purified NGF injected into the synovium of normal rat joints did not induce knee joint inflammation, suggesting that NGF does not play a causative role in arthritis (Aloe et al., Growth Factors 9: 149-155 (1993)).

High levels of NGF are associated with allergic inflammation, which has been suggested to be associated with mast cell degranulation (Bonini et al., Proc. Natl. Acad. Sci. USA 93: 10955-10960 ( 1996)).
Elevated NGF levels have also been observed in both allergic and non-allergic asthma (Bonini et al., Supra). Mast cells, eosinophils, and T-lymphocytes have all been proposed to contribute to this inflammatory disease, and the correlation between NGF serum levels and total IgE antibody titers indicates that NGF is inflammatory This suggests that it contributes to the immune response. In both mice and humans, airway inflammation induced by allergens has been associated with local production of NGF (Braun et al., Int. Arch. Allergy Immunol. 118: 163-165 (1999)).

NGF has been shown to control the development of the increased airway hyperresponsiveness characteristic of bronchial asthma (Braun et al., Eur. J. Immunol. 28: 3240-3251 (1998)). In fact, in one study, treatment of allergen-sensitized mice with anti-NGF antibodies prevented the development of airway hypersensitivity after local allergen challenge (Braun et al., Int. Arch. Allergy Immunol. 118: 163 -165 (1999)).
Despite the promising results obtained in mice, the reported side effects of neutralizing anti-NGF antibodies to the immune system are anti-NGF as a therapy in the prevention or treatment of asthma or other diseases or conditions in human patients. A serious question was raised about the possibility of using antibodies. In particular, Torcia et al., Cell 85: 345-356 (1996) identified NGF as an autocrine survival factor for memory B lymphocytes, and in vivo administration of neutralizing anti-NGF antibody in mice resulted in memory B cell It has been shown to cause depletion and extinguish secondary antigen-specific immune responses. Garaci et al., Proc. Natl. Acad. Sci. USA 96: 14013-14018 (1999) report that NGF is an autocrine survival factor that rescues human monocytes / macrophages from cytopathic effects of HIV infection. ing. This report, along with the findings of Torica et al., Supra, may suggest that anti-NGF antibodies have the potential to compromise the immune system of treated subjects.

(Outline of the present invention)
The present invention is based on the unexpected discovery that in vivo administration of a therapeutically effective amount of an anti-NGF monoclonal antibody (antibody 911) had no side effects on the immune system of an experimental allergic mouse model. Yes. Accordingly, antibodies associated therewith are quite promising in the treatment of NGF-related diseases including asthma in human patients.
In one aspect, the invention provides that antibodies have no significant side effects on the patient's immune system, bind hNGF with nanomolar affinity, and inhibit hNGF binding to human TrkA (hTrkA) in vivo. It relates to a method of controlling NGF-related diseases in human patients by administering to the patient an effective amount of a possible anti-human NGF (anti-hNGF) monoclonal antibody.

In one embodiment, the binding affinity of the antibody to hNGF is preferably about 0.10 to about 0.80 nM, more preferably about 0.15 to about 0.75 nM, and even more preferably about 0.18 to About 0.72 nM.
In other embodiments, the antibody binds essentially the same hNGF epitope as the antibody selected from the group consisting of MAb911, MAb912 and MAb938, more preferably the same epitope as MAb911.
In yet other embodiments, the antibody can cross-react with mouse NGF (muNGF).
The antibody is also selected from the group consisting of antibody fragments, preferably Fab, Fab ′, F (ab ′) ₂ , Fv fragments, diabodies, single chain antibody molecules and multispecific antibodies formed from antibody fragments. Antibody fragments, and more preferably single chain Fv (scFv) molecules.
In other embodiments, the antibody is chimeric. It can also be humanized or human.
In yet other embodiments, the antibody is bispecific. Bispecific antibodies will have anti-IgE specificity.
A controlled NGF-related disease is preferably not associated with the action of NGF on the nervous system.

In one embodiment, the NGF-related disease is an inflammatory condition, preferably selected from the group consisting of asthma, arthritis, multiple sclerosis, lupus erythematosus and psoriasis.
In a preferred embodiment, the symptom is asthma. In other embodiments, the symptom is arthritis, preferably rheumatoid arthritis. In yet other embodiments, the symptom is psoriasis.
In yet other embodiments, the antibody is administered in combination with other therapeutic agents for the treatment of inflammatory conditions. Thus, the antibody may be administered in combination with other therapeutic agents for the treatment of asthma. In one embodiment, the antibody is administered with a corticosteroid, preferably beclomethasone dipropionate (BDP). In other embodiments, the antibody is administered with an anti-IgE antibody, such as rhuMAb-E25 or rhuMAb-E26. For the treatment of rheumatoid arthritis, the antibody may be administered in combination with an anti-TNF antibody, or an antibody or immunoadhesin that specifically binds to a TNF receptor.

In other aspects, the present invention is capable of binding hNGF in vivo and in combination with a pharmaceutically acceptable carrier with an affinity in the nanomolar range and inhibiting the binding of human TrkA and hNGF. It relates to a pharmaceutical composition comprising a chimeric, humanized or human anti-human NGF monoclonal antibody, which antibody has no significant side effects on the patient's immune system. The antibody in the pharmaceutical composition is composed of antibody fragments, preferably Fab, Fab ′, F (ab ′) 2, Fv fragments, diabodies, single chain antibody molecules formed from antibody fragments and multispecific antibodies. An antibody fragment selected from the group consisting of:
In one embodiment, the antibody is a dual property antibody. The dual property antibody may be capable of specifically binding to natural human IgE or natural human TNF or natural human TNF receptor.
In other embodiments, the pharmaceutical composition further comprises other pharmaceutically active ingredients, such as ingredients suitable for the treatment of inflammatory conditions. The inflammatory condition is preferably one selected from the group consisting of asthma, multiple sclerosis, arthritis, lupus erythematosus and psoriasis. In one embodiment, the inflammatory condition is asthma. In other embodiments, the inflammatory condition is arthritis, preferably rheumatoid arthritis. In other embodiments, the inflammatory condition is psoriasis.

In other aspects, the present invention is capable of binding hNGF in vivo and in combination with a pharmaceutically acceptable carrier with an affinity in the nanomolar range and inhibiting the binding of human TrkA and hNGF. Containers comprising chimeric, humanized or human anti-human NGF monoclonal antibodies, articles of manufacture comprising pharmaceutical compositions, and instructions for using the compositions to control NGF-related diseases in human patients The antibody has no significant side effects on the patient's immune system.
In one embodiment, the article of manufacture preferably comprises further pharmaceutically active agents suitable for the treatment of inflammatory conditions. This inflammatory condition is preferably selected from the group consisting of asthma, multiple sclerosis, arthritis, lupus erythematosus and psoriasis.

Detailed Description of Preferred Embodiments
A. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd edition., J. Wiley & Sons (New York, New York 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, New York 1989). See Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, used in the practice of the present invention. Indeed, the present invention is not limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

As used herein, the terms “nerve growth factor” and “NGF” are defined as native sequence NGF of all mammalian species, including humans.
“NGF receptor” refers to a polypeptide that is bound or activated by NGF. NGF receptors include TrkA and p75 receptors of all mammalian species, including humans.

  The term “native sequence” in the context of NGF or other polypeptide refers to a polypeptide having the same amino acid sequence as the corresponding polypeptide obtained from nature, regardless of the method of its preparation. Such native sequence polypeptides can be isolated from nature and can be produced by recombinant and / or synthetic methods or any combination thereof. The term “native sequence” specifically refers to naturally occurring truncated or secreted forms (eg, extracellular domain sequences), naturally occurring variants (eg, alternative splicing), and naturally occurring allelic variants of full-length polypeptides. Includes.

  “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity for a particular antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. The latter type of polypeptide is produced, for example, at low levels by the lymphatic system and at increased levels by myeloma.

  “Natural antibodies and natural immunoglobulins” are heterotetrameric glycoproteins of approximately 150,000 daltons, usually composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds in different immunoglobulin isotype heavy chains is different. Each heavy and light chain also has intrachain disulfide bridges between regularly spaced sites. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the light chain constant domain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is the heavy chain Aligned with the variable domain. Certain amino acid residues are thought to form an interface between light and heavy chain variable domains (Chothia et al., J. Mol. Biol. 186: 651 [1985]; Novotny and Haber, Proc. Natl. Acad Sci. USA 82: 4592 [1985]; Chothia et al., Nature 342: 877-883 [1989]).

  The term “variable” refers to the fact that a site in a variable domain varies widely in sequence within an antibody and is used for the binding and specificity of each particular antibody for its particular antigen. . However, variability is not evenly distributed across the variable domains of antibodies. It is concentrated in three segments called hypervariable regions or complementarity determining regions (CDRs) of both the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR). The natural heavy and light chain variable domains each take a predominantly β-sheet configuration, which forms a loop connecting the β-sheet structures and in some cases three CDRs that form part of it. Contains four FR regions connected by The CDRs of each chain are more closely linked to the FR, and together with the CDRs of the other chains, contribute to the formation of the antibody antigen binding site (see Kabat et al., (1991)). The constant domain is not directly related to the binding of the antibody to the antigen, but indicates the involvement of the antibody in various effector functions, such as antibody-dependent cytotoxic activity.

Antibody papain digestion yields two identical antigen-binding fragments, each called a “Fab” fragment with a single antigen-binding site, and a residue “Fc” fragment indicating that the name can be easily crystallized. . Pepsin treatment yields an F (ab ′) ₂ fragment that has two antigen linking sites and is still capable of cross-linking antigen.

  “Fv” is the minimum antibody fragment which has a complete antigen recognition and binding site. In the double-chain Fv species, this region consists of a dimer of one heavy and one light chain variable domain with tight non-covalent bonds. In a single chain Fv species, one heavy and one light chain variable domain is such that the light and heavy chains can associate as a “dimer” structure that is similar in terms of the double chain Fv species. It can be covalently linked by an adaptive peptide linker. It is in this structure that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. To be precise, the six CDRs confer antigen binding specificity to the antibody. However, even with a lower affinity than the entire binding site, even a single variable domain (or half of an Fv containing only three CDRs specific for an antibody) has the ability to recognize and bind to an antigen. Have.

The Fab fragment also has the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab ′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the antibody hinge region. Fab′-SH is a symbolic notation here for Fab ′ in which the cysteine residue of the constant domain has a free thiol group. F (ab ′) ₂ antibody fragments were originally produced as pairs of Fab ′ fragments that have hinge cysteines. Other chemical couplings of antibody fragments are also known.

The “light chain” of an antibody (immunoglobulin) from any vertebrate species can be applied to one of two distinctly different types, termed κ and λ, based on the amino acid sequence of its constant domain.
Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these are further subclasses (isotypes), eg, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The different classes of subunit structures and three-dimensional structures of immunoglobulins are well known.

The term “antibody” specifically includes monoclonal antibodies comprising antibody fragment clones.
“Antibody fragments” comprise a portion of an intact antibody, usually the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ and Fv fragments; diabodies; single chain antibody molecules comprising single chain Fv (scFv) molecules; and duplexes formed from antibody fragments Specific antibodies are included.

  The term “monoclonal antibody” as used herein refers to an antibody (or antibody fragment) obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies that make up the population are naturally present in small amounts. Identical except for possible mutations that occur. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that generally include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are free from contamination by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not meant to imply that production of the antibody by any particular method is required. . For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described in Kohler et al., Nature, 256: 495 [1975] or may be recombinant DNA methods (eg, US Pat. No. 4,816,567). You may create it by reference. “Monoclonal antibodies” can also be obtained using techniques described in, for example, Clackson et al., Nature, 352: 624-628 [1991] and Marks et al., J. Mol. Biol., 222: 581-597 (1991). , Including antigen recognition and binding site clones, including antibody fragments (Fv clones) isolated from phage antibody libraries.

  Here, monoclonal antibodies include in particular “chimeric” antibodies (immunoglobulins), which are antibodies of which part of the heavy and / or light chain is derived from a particular species or belongs to a particular antibody class or subclass. Antibodies that are identical or homologous to the corresponding sequences, but the rest of the chain are derived from other species or belong to other antibody classes or subclasses, as long as they exhibit the desired biological activity Identical or homologous to the corresponding sequences of the antibody fragments (US Pat. No. 4,816,567 to Cabililly et al .; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 [1984]).

“Humanized” forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab ′, etc.) that contain minimal sequence derived from non-human immunoglobulin. F (ab ′) ₂ or other antigen-binding sequence of the antibody). For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which the recipient's complementary chain determining region (CDR) residues have the desired specificity, affinity, and capacity. Alternatively, it is substituted with a residue derived from a CDR (donor antibody) of a species other than human such as rabbit. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the grafted CDR or frame sequences. These modifications are made to further refine and optimize antibody performance. Generally, a humanized antibody has at least one, typically all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are of human immunoglobulin sequences, typically It will contain substantially all of the two variable domains. An optimal humanized antibody will also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). And Clark, Immunol. Today 21: 397-402 (2000). Humanized antibodies include Primatized (trade name) antibodies derived from antibodies produced by macaque monkeys that have been immunized with the antigen of interest of the antigen binding region of the antibody.

  “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In general, scFv polypeptides further comprise a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For an overview of scFv, see Pluckthun, Dall'Acqua and Carter, Curr. Opin. Struct in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994). Biol. 8: 443-450 (1998) and Hudson, Curr. Opin. Immunol. 11: 548-557 (1999).

  The term `` diabody '' refers to a small antibody fragment with two antigen binding sites, which fragment is in the same polypeptide chain (VH-VL) to the light chain variable domain (VL) to the heavy chain variable domain (VH ) Are combined. Using a linker that is so short that it cannot pair two domains on the same chain, it forces the domain to pair with the complementary domain of the other chain to form two antigen-binding sites . Diabodies are described in further detail, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

  An “isolated” antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminants in the natural environment are substances that interfere with the use of antibodies for diagnosis or therapy, and include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is (1) at least 95% by weight or more preferably 99% by weight antibody as determined by the Lowry method, and (2) by using a spinning cup sequenator. To the extent sufficient to obtain 15 N-terminal or internal amino acid sequence residues, or (3) SDS-PAGE under non-reducing or reducing conditions, and uniformly by Coomassie blue or preferably silver staining It can be purified until. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

  By “neutralizing antibody” is meant an antibody molecule that can eliminate or significantly reduce the effector function of the target antigen to which it binds. Thus, “neutralizing” anti-NGF antibodies can eliminate or significantly reduce effector function, eg, NGF receptor binding and / or induction of cellular responses. A “significant” decrease is at least about 60%, preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%, even more preferably a target antigen (eg, NGF) effector function. Means a reduction of at least about 85%, most preferably at least about 90%.

An antibody is capable of “inhibiting binding” of a ligand to a receptor if it produces an objectively measurable decrease in the ability of the ligand to bind to the receptor.
The term “epitope” is used to refer to the binding site of an antibody (monoclonal or polyclonal) on a protein antigen.

  Antibodies that bind to a particular epitope can be identified by “epitope mapping”. Many methods are known in the art for mapping and characterizing epitope locations on proteins, including, for example, Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Analyzing the crystal structure of antibody-antigen complexes, as described in Chapter 11 of New York, 1999, competition assays, gene fragment expression assays, and synthetic peptide-based assays. Competitive assays are discussed below. According to the gene fragment expression assay, the open reading frame encoding the protein is fragmented either randomly or by a specific genetic construct, and the activity of the expressed fragment of the protein with the antibody being tested is measured. Is done. For example, gene fragments are produced by PCR and then transcribed in the presence of radioactive amino acids and translated into proteins in vitro. Binding of the antibody to the radiolabeled protein fragment is then measured by immunoprecipitation and gel electrophoresis. Specific epitopes are also identified by using a large library of random peptide sequences displayed on the surface of phage particles (phage library). Alternatively, the identified library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. The latter method is suitable for identifying linear epitopes of about 5 to 15 amino acids.

  When two antibodies recognize the same or sterically overlapping epitopes, the antibodies bind to “essentially the same epitope” as the reference antibody. To determine whether two epitopes bind to the same or sterically overlapping epitopes, the most widespread and rapid method is a competitive assay, which uses either labeled antigen or labeled antibody, all Can be configured in different formats. Usually, the antigen is immobilized on a 96 well plate and the ability of the unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive or enzyme label.

  The term amino acid or amino acid residue as used herein refers to a naturally occurring L amino acid or D amino acid, as further described below for variants. One and three letter abbreviations widely used for amino acids are used here (Bruce Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (3rd edition. 1994)).

  A “variant” is an antibody that retains the same biological activity while differing in some respect from a natural antibody. A variant may have an amino acid sequence that differs from that of a native antibody as a result of the insertion, deletion, modification, and / or substitution of one or more amino acid residues within the native sequence. A variant may have a glycosylation pattern that differs from that of a natural antibody. Furthermore, the variant may be a covalently modified natural antibody.

  A “disorder” is any condition that would benefit from the treatment described in the present invention. “Disease” and “Symptoms” are used interchangeably herein and include chronic and acute diseases or conditions, including conditions that make a mammal susceptible to infection with the disease in question. Non-limiting examples of diseases to be treated here include lupus erythematosus, contact dermatitis, eczema, herpes zoster, postherpetic neuralgia, hyperalgesia, chronic pain, irritable bowel syndrome, Crohn's disease, colitis, cystitis Including arthritis, including multiple sclerosis, asthma, psoriasis, and chronic arthritis and rheumatoid arthritis. Preferred diseases to be treated by the present invention are inflammatory conditions such as asthma, multiple sclerosis, arthritis, lupus erythematosus and psoriasis.

  “Inflammatory symptoms” are associated with symptoms characterized by one or more of pain, fever, redness, swelling and loss of function, and tissue injury, infection, hypersensitivity or injury.

  The term “disease state” refers to the physiological state of a cell or whole mammal in which a disorder, cessation, or abnormality of the cell or body functional system, or organ is occurring.

  The term “effective amount” or “therapeutically effective amount” refers to an amount of an agent effective to treat and / or prevent a mammalian disease, or disease or undesirable physiological condition. In the present invention, an “effective amount” of an anti-NGF antibody prevents, reduces, blunts, or delays the onset of a disease such as lupus, multiple sclerosis, asthma, psoriasis or arthritis; lupus, multiple sclerosis, asthma One or more symptoms associated with such a disease to a certain extent; and / or to reduce, prevent or inhibit the development of a disease such as psoriasis or arthritis; Can be reduced.

  In the methods of the present invention, the term “control” and grammatical variants thereof prevent, partially or completely inhibit, reduce, delay or reduce physiological symptoms such as inflammatory responses associated with unfavorable symptoms such as asthma. Used to refer to blunting.

  “Treatment” or “treating” as used herein refers to curative therapy, prophylactic therapy, and preventative therapy. Those in need of treatment include those already suffering from the disease as well as those in which the disease is being prevented that are susceptible to the disease. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, disappearance of disease range, stable disease (ie Conditions that do not worsen), delay or slowing of disease progression, amelioration or reduction of disease state, and remission (either partial or whole). “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment along with those prone to having symptoms or diseases or those in which symptoms or diseases are prevented include those already having symptoms or diseases.

  A “significant side effect” to the immune system is an action that compromises the immune system and / or inhibits the normal immune response to antigenic stimulation. An example of significant side effects on the immune system would be a reduced humoral immune response.

  A “pharmaceutically acceptable” carrier, excipient, or stabilizer is one that is nontoxic to the cells or mammals exposed to it at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers are buffers such as phosphate, citrate, and other organic acid salts; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin, or immunoglobulins; hydrophobic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin Chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN (trade name), polyethylene glycol (PEG), and Includes PLURONICS (trade name).

  “Liposomes” are small vesicles composed of various types of lipids, phospholipids and / or surfactants, and are used for drugs to mammals (eg, the anti-NGF antibodies disclosed herein and the context). Accordingly, it is useful for the delivery of chemotherapeutic agents). The components of the liposome are usually arranged in a bilayer format similar to the lipid arrangement of biological membranes.

  The term “package insert” is used to mean instructions that are usually included in commercial packages of therapeutic products, including information about efficacy, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. Is done.

  “Mammal” for the subject of treatment means any animal classified as a mammal, including humans, household and agricultural animals, zoos, sports, or pet animals such as dogs, horses, cats, cows, etc. To do. Preferably the mammal is a human.

B. Methods of practicing the invention As described in more detail below, administration of anti-NGF antibody 911 in a mouse model of asthma reduced the degree of airway hypersensitivity and inflammation but reduced total immunoglobulin levels and IgE. It did not reduce the humoral immune response to the inhaled antigen as measured by serum levels.
Anti-NGF antibodies are known in the art. Anti-NGF antibodies useful in the present invention include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, heteroconjugate antibodies, and antibody fragments, and glycosylated variants of antibodies. And modified antibodies, including amino acid sequence variants of the antibody and covalently modified antibodies. This antibody can be produced by any method known in the art.
Thus, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (US Pat. No. 4,816,567).

  In summary, in the hybridoma method, mice or other suitable host animals, such as hamsters or macaques, are immunized as described above to produce or produce antibodies that specifically bind to the protein used for immunization. Induce lymphocytes that can Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and fused with a myeloma cell line using an appropriate fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pages 59-103). [Academic Press, 1986]). The hybridoma cells thus prepared are seeded and grown in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of unfused, parental myeloma cells. For example, parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), and hybridoma media normally inhibit the growth of HGPRT-deficient cells such as hypoxanthine, aminopterin and thymidine (HAT media). Containing substances to be used. A preferred myeloma cell line is a mouse myeloma line, such as the MOP-21 and MC-11 mouse tumors available from Souk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 or X63-Ag8-653 cells available from American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, 51 -63, (Marcel Dekker, Inc., New York, 1987)). The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is measured by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of a monoclonal antibody can be measured, for example, by Scatchard analysis as described in Munson et al., Anal. Biochem., 107: 220 (1980). Once a hybridoma cell producing an antibody with the desired specificity, affinity, and / or activity is identified, it can be subcloned by limiting dilution and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, 59-103 (Academic Press, 1986)). Monoclonal antibodies secreted by subclones are appropriately separated from medium, ascites fluid, or serum by routine antibody purification methods such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Is done.

  Recombinant production of antibodies requires the isolation of DNA encoding the antibody or antibody chain. The DNA encoding the monoclonal antibody is readily separated using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody), To be sequenced. Hybridoma cells are a preferred source of such DNA. Once isolated, this DNA is placed into an expression vector in order to obtain monoclonal antibody synthesis in a recombinant host cell, which is then transformed into an E. coli cell that does not produce any immunoglobulin protein in this context, monkey COS. The cells may be transfected into host cells such as Chinese hamster ovary (CHO) cells or myeloma cells. For example, by replacing homologous mouse sequences with coding sequences for human heavy and light chain constant domains (Morrison et al., Proc. Nat. Acad. Sci., USA, 81: 6851 [1984]) or immune This DNA can be modified by covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide to the globulin coding sequence. In this method, “chimeric” or “hybrid” antibodies are prepared to have the binding specificity of the anti-NGF monoclonal antibodies herein.

  Typically, such non-immunoglobulin polypeptides replace the constant region of an antibody or replace the constant region of one antigen binding site of an antibody of the invention and have specificity for an NGF polypeptide. A chimeric bivalent antibody is created that contains one antigen binding site and another antigen binding site with specificity for a different target such as a high affinity IgE receptor or a TNF receptor. Chimeric or hybrid antibodies can also be prepared in vitro using methods known in synthetic protein chemistry, including those related to cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by formation of a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyridimidate.

  Non-human, eg mouse antibodies, can be humanized. In general, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as “import” residues from the “import” variable domain. Humanization is basically performed by Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science. 239: 1534-1536 (1988)) by substituting the corresponding sequence of the human antibody with the CDR or CDR sequence of the rodent.

  It is important that antibodies be humanized with retention of high affinity for antigens and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of parental sequences and conceptual humanized products using a three-dimensional model of the parental and humanized sequences. Three-dimensional immunoglobulin models are widely available and are well known to those skilled in the art. Computer programs are available that exemplify and display possible three-dimensional configuration structures of selected candidate immunoglobulin sequences. Examination of these representations allows analysis of residues that affect the potential role of residues in the functionalization of candidate immunoglobulin sequences, ie, the ability of candidate immunoglobulins to bind to their antigens. In this way, FR residues can be selected and combined from consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. See US Pat. No. 5,821,337 for further details.

The present invention also includes human anti-NGF antibodies. As noted above, such human antibodies can be made by hybridoma methods using human myeloma or mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies (eg, Kozbor, J. Immunol 133, 3001 (1984), and Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, (1987)). It is now possible to create transgenic animals (eg, mice) capable of producing a repertoire of human antibodies without the production of immunoglobulins, eg, antibody heavy chain J in chimeric and germline mutant mice. the homozygous deletion region (J _H) gene, the complete inhibition of endogenous antibody production occurs have been explained. human germline into such germ mutant mice Transposition of the epithelial globulin gene array results in the production of human antibodies upon antigen stimulation, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-2555 (1993); (1993) See also Mendez et al. (Nat. Genet. 15: 146-156 (1997)) for an improved version of this technique.

  Alternatively, phage display technology (MaCafferty et al., Nature 348, 552-553 [1990]) produces human antibodies and antibody fragments in vitro from the immunoglobulin variable (V) domain gene repertoire of unimmunized donors. Can be used to According to this technique, antibody V domains are cloned in-frame into either the major or minor coat protein gene of a filamentous bacteriophage such as M13 or fd and displayed as functional antibody fragments on the surface of the phage particle. . Since filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of genes encoding antibodies that exhibit these properties. Thus, phage mimics some properties of B cells. Phage display can be performed in a variety of formats; see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3, 564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature 352, 624-628 (1991) isolated a variety of anti-oxazolone antibodies from a small random combinatorial library of V genes removed from the spleens of immunized mice. A repertoire of non-immunized human donor V genes can be constructed, and antibodies against a variety of antigens (including self-antigens) can be generated essentially by Marks et al., J. Mol. Biol. 222, 581-597 ( 1991), or Griffith et al., EMBO J. 12, 725-734 (1993). In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes introduced give higher affinity and B cells displaying high affinity surface immunoglobulin preferentially replicate and differentiate during subsequent antigen stimulation. This natural process can be mimicked using a technique known as “chain shuffling” (Marks et al., Bio / Technol. 10, 779-783 [1992]). In this method, the affinity of the “primary” human antibody obtained by phage display is compared to the naturally occurring variant of the V domain gene (repertoire) obtained from a donor that has not immunized heavy and light chain V region genes. ) Repertoire over time can be improved. This technique allows the production of antibodies and antibody fragments with an affinity on the order of nM. A strategy for making a fairly large phage antibody repertoire (also known as “under all libraries”) is described by Waterhouse et al., Nucl. Acids Res. 21, 2265-2266 (1993), The isolation of high affinity human antibodies directly from such large phage libraries has been reported by Griffith et al., EMBO J. 13: 3245-3260 (1994) in print.

  Gene shuffling is also used to derive human antibodies from rodent antibodies, which have similar affinities and specificities for the initial rodent antibody. According to this method, also called “epitope imprinting”, the light chain V domain gene of a rodent antibody obtained by phage display technology is replaced with a repertoire of human V domain genes to create a rodent-human chimera. Selection with an antigen results in the isolation of a human variable region that can use a functional antigen binding site, ie, the epitope dominates the choice of partner (imprint). When the process is repeated to replace the remaining rodent V domain, human antibodies are obtained (see PCT patent application WO 93/06213 issued April 1, 1993). Unlike traditional humanization of rodent antibodies by CDR grafting, this technique provides fully human antibodies that do not have a framework or CDR residues of rodent origin.

  The present invention specifically includes bispecific antibodies. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs with different specificities of the two heavy chains (Millstein and Cuello, Nature, 305: 537-539 (1983)). By different and more preferred techniques, antibody variable domains with the desired binding specificity (antibody-antigen combining site) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is desirable to have a first heavy chain constant region (CH1) containing the site present in the light chain bond that is present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and cotransfected into a suitable host organism. This provides a high degree of flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments where the unequal proportions of the three polypeptide chains used in the construction provide the highest production. However, if high production occurs due to expression of at least two polypeptide chains in equal proportions, or if the proportion is not particularly significant, the coding of all two or three polypeptide chains into one expression vector Can be inserted. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

  Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies are used, for example, to target immune system cells to unwanted cells [US Pat. No. 4,676,980] and for the treatment of HIV infection [WO 91/00360; WO 92/200373; Europe. Patent No. 03089] has been proposed. Heteroconjugate antibodies can be made using any conventional cross-linking method. Suitable crosslinking agents are known in the art and are disclosed in US Pat. No. 4,676,980, along with a number of crosslinking techniques.

Antibody fragments have been traditionally derived by proteolytic digestion of intact antibodies (e.g. Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229: 81). (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, Fab′-SH fragments can be recovered directly from E. coli and chemically combined to form F (ab ′) ₂ fragments (Carter et al., Bio / Technology 10: 163-167 [1992 ]). In other embodiments, leucine zipper GCN4 is used to facilitate assembly of F (ab ′) ₂ molecules to form F (ab ′) ₂ fragments. According to other techniques, Fv, Fab or F (ab ′) ₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled artisan.

  For certain embodiments of the invention, it may be desirable to modify the antibody fragment to increase serum half-life. This can be accomplished, for example, by introducing a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment, or then at either end or center of the antibody fragment, e.g., DNA or peptide synthesis). By introducing an epitope into the peptide tag fused by See International Publication No. 96/32478, issued on October 17, 1996.

Salvage receptor binding epitopes generally constitute a region in which one or more amino acid residues of one or two loops of the Fc domain are transferred to similar positions in an antibody fragment. Even more preferably, three or more residues of one or two loops of the Fc domain are transferred. Even more preferably, the epitope is taken from the CH2 domain of the Fc region (eg of IgG) and transferred to the CH1, CH3, or _VH region of the antibody, or one or more such regions. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the C _L region or V _L region, or both, of the antibody fragment.

  Amino acid sequence variants containing specifically disclosed anti-NGF antibody substitutions, insertions, and / or deletions are prepared by introducing appropriate nucleotide changes into the encoded DNA, or by peptide synthesis. Methods for making such variants are well known in the art and include, for example, “alanine scanning mutagenesis” as described in Cunningham and Wells Science, 244: 1081-1085 (1989). Certain types of amino acid variants of the antibody alter the original glycosylation pattern of the antibody. By altering means removing one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody.

Screening for Antibodies with Desired Properties Antibodies useful in the present invention neutralize NGF activity. Thus, for example, a neutralizing anti-NGF antibody of the present invention can be identified by incubating NGF with a candidate antibody and monitoring the neutralization of binding and biological activity of NGF. Binding assays can be performed with purified NGF polypeptide or cells that naturally express or are transfected to express NGF polypeptide. In one embodiment, the binding assay is a competitive binding assay that evaluates the ability of a candidate antibody to compete with a known anti-NGF antibody for NGF binding. The assay can be performed in a variety of formats, including ELISA types.

  The ability of a candidate antibody to neutralize the biological activity of NGF is demonstrated by NGF-mediated survival in the dorsal root ganglion survival bioassay of fetal rats, for example, as described in Hongo et al. This is done by monitoring the ability of the candidate antibody to inhibit.

  For the screening of antibodies that bind to epitopes on NGF bound by the antibody of interest, routine crosses described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Blocking assays can be performed. Alternatively, or in addition, epitope mapping can be performed by methods known in the art. For example, NGF epitopes bound by the monoclonal antibodies of the invention can be measured by competitive binding analysis as described in Fendly et al. Cancer Research 50: 1550-1558 (1990). Cross-blocking studies can be performed by direct fluorescence emission on intact cells using PANDEX Screen Machine, which quantifies fluorescence emission. This method is conjugated with fluorescein isothiocyanate (FITC) using established techniques (Wofsy et al., Selected Methods in Cellular Immunology, p. 287, Mishel and Schiigi (ed.) San Francisco: WJ Freeman Co. (1980)). NGF-expressing cells and purified monoclonal antibody in suspension are added to a PANDEX (trade name) plate well and incubated, and fluorescence emission is quantified by PANDEX (trade name). A monoclonal antibody is considered to share an epitope if it blocks 50% or more compared to an unrelated monoclonal antibody control, each blocking other bindings.

Anti-NGF antibodies useful in the present invention can also be identified using combinatorial libraries for synthetic antibody clones having the desired activity or action. Such methods are known in the art. In summary, synthetic antibody clones are produced by phage libraries containing phage that display various fragments of antibody variable regions (Fv) fused to phage coat proteins (eg, Fab, F (ab ′) ₂ ,...). Select. Such phage libraries are picked up by affinity chromatography against the desired antigen. Clones that express Fv fragments capable of binding to the desired antigen are absorbed into the antigen and are thus separated from unbound clones in the library. The binding clones are then eluted from the antigen and can be further enriched by further antigen absorption / elution cycles. Appropriate anti-NGF antibodies for use in the present invention can be designed by designing a suitable antigen screening method to select a phage clone of interest, the Fv sequence of the subject phage clone and the appropriate constant region (Fc ) To obtain a full-length anti-NGF antibody clone using the sequence.

  Results obtained with cell-based biological assays can then be followed by testing in animals, eg mice, models, and human clinical trials. If desired, mouse monoclonal antibodies identified as having the desired properties are converted to chimeric antibodies by techniques known in the art, including “gene conversion mutations” described in US Pat. No. 5,821,337. Or humanized.

C. Pharmaceutical preparations The therapeutic preparations of antibodies used in the present invention comprise antibodies having the desired degree of purity, in the form of lyophilized preparations or aqueous solutions, the optimal pharmaceutically acceptable carrier, excipient or Prepared and stored by mixing with stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and buffers such as phosphate, citrate, and other organic acids; ascorbic acid and methionine Antioxidants containing; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclo Hexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; glycine, glutamine, Amino acids such as sparagin, histidine, arginine, or lysine; monosaccharides, disaccharides including glucose, mannose, or dextrin, other carbohydrates, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol; sodium etc. Non-ionic surfactants such as metal complexes (eg, zinc-protein complexes) and / or TWEEN (trade name), PLURONICS (trade name), and polyethylene glycol (PEG) Contains agents.

  Neutralized anti-NGF antibodies useful in the methods of the invention can also be formulated as immunoliposomes. Liposomes containing antibodies are described, for example, in Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); Prepared by methods known in the art as described in US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in US Pat. No. 5,013,556.

  Particularly useful liposomes can be made by the reverse phase evaporation method with a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter with a defined pore size to obtain liposomes having a desired diameter. Fab ′ fragments of the antibodies of the invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982). . In some cases, a chemotherapeutic agent (eg, doxorubicin) is included within the liposome. See Gabizon et al., J. National Cancer Inst. 81 (19) 1484 (1989).

  The active ingredient can also be used in colloidal drug delivery systems (eg, in microcapsules prepared by coacervation techniques or by interfacial polymerization, eg, hydroxymethylcellulose or gelatin-microcapsules and poly (methyl methacrylate) microcapsules, respectively. , Liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules) or macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Science 16th edition, Osol, A. Ed. (1980).

  Sustained release formulations may be prepared. Suitable examples of sustained release formulations include a semi-permeable matrix of solid hydrophobic polymer containing antibodies, which matrix is in the form of a molded article, such as a film or microcapsule. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide (US Pat. No. 3,773,919), L-glutamic acid and ethyl- Degradable lactic acid-glycolic acid copolymer such as L-glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT (trade name) (injectable globules of lactic acid-glycolic acid copolymer and leuprolide acetate), poly- (D)- (-)-3-Hydroxybutyric acid is included.

  The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide antibodies that bind to different epitopes of NGF or NGF receptor in one formulation. Alternatively or additionally, the composition can further comprise other bioactive compounds such as anti-inflammatory agents. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

  The formulation used for in vivo administration should be sterile. This is easily accomplished by filtration through sterile filtration membranes.

  The therapeutic anti-NGF antibody composition is generally placed in a container equipped with a sterile access port, for example, an intravenous solution bag or vial having a stop pierceable by a hypodermic needle.

D. Treatment with anti-NGF antibodies According to the present invention, it may be possible to treat various diseases or conditions using anti-NGF antibodies. Exemplary symptoms or diseases include asthma, psoriasis and arthritis. Anti-NGF antibodies can be used to prevent the onset of the active stage of the disease, treat the symptoms commonly experienced, and treat the potential disease itself.

  Despite advances in understanding the cellular and molecular mechanisms that control allergic responses and improved therapies, the incidence of allergic diseases, particularly asthma, has increased dramatically in recent years (Beasley et al., J. Allergy Clin. Immunol. 105: 466-472 (2000); Peat and Li, J. Allergy Clin. Immunol. 103: 1-10 (1999)). Allergic diseases can be treated, for example, by allergen-based vaccination given by increasing allergens by injection over several years. Mild asthma can usually be controlled by relatively low doses of inhaled corticosteroids in most patients, while moderate asthma is usually associated with inhaled long-chain β-antagonists or leukotriene inhibitors Treated by further administration. However, the treatment of some asthma is still a serious medical problem. Anti-IgE antibodies (rhuMAb-E25, Xolair (trade name) Genentech, Inc., Tanox, Inc. and Novartis Pharmaceuticals Co., Ltd.), which are currently pending FDA approval, are allergic asthma and seasonal allergies While showing promising results for the initial therapeutic intervention of conditions that cause rhinitis symptoms, there is a need for further therapeutic strategies and drug development to control allergic diseases such as asthma.

  The anti-NGF antibodies of the present invention may typically be used to treat asthma characterized by the development of cough, wheezing, chest pain, and / or dyspnea, and other diseases associated with airway hypersensitivity. it can.

  The anti-NGF antibody of the present invention may be used to treat other inflammation such as multiple sclerosis, colitis, inflammatory bowel disease, cystitis, eczema, contact dermatitis, arthritis including chronic arthritis and rheumatoid arthritis, Crohn's disease and psoriasis. It is also useful for symptom management.

  In addition, anti-NGF antibodies are useful for the treatment of other diseases that may be associated with increased levels of NGF including, for example, lupus erythematosus, herpes zoster, postherpetic neuralgia, hyperalgesia, chronic pain.

  The anti-NGF antibody is intramuscularly, intraperitoneally, intracerebral spinal, subcutaneous, intraarticular, intraosseous, arachnoid by well-known methods such as intravenous administration as a bolus or continuous infusion over a period of time. It is administered to a mammal, preferably a human patient, by intracavitary, oral or topical route. Anti-NGF antibodies are also administered by inhalation. Nebulizers for commercially available liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. The liquid formulation can be atomized directly and the lyophilized powder can be atomized after reconstitution. Alternatively, the anti-NGF antibody can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder. For the treatment of other symptoms characterized by airway hypersensitivity and asthma, the preferred route of administration is inhalation.

  Other therapies may be in combination with administration of anti-NGF antibodies. Preferably, there is a period in which both (or all) active agents simultaneously exert their biological activity, and the combined administration includes simultaneous administration using separate formulations or a single pharmaceutical formulation, and continuous in any order Administration is included. For the treatment of asthma, anti-IgE antibodies, particularly rhuMAb-E25 (Xolair (trade name)), or the second generation antibody molecule rhuMAb-E26 (Genentech, Inc.) There will be particular advantages to using. The rhuMAb-E25 antibody is a recombinant humanized anti-IgE monoclonal antibody developed to intervene early in the allergic process. Combination applications also include the possibility of administering two antibodies in a single pharmaceutical formulation, or using dual-characteristic antibodies with anti-NGF and anti-IgE specificity. In another preferred embodiment, the anti-NGF antibody herein is administered in combination with an inhaled corticosteroid such as beclomethasone dipropionate (BDP) treatment. For the treatment of rheumatoid arthritis or Crohn's disease, the antibodies of the invention can be administered in combination with other therapies known to treat these conditions. For example, anti-NGF antibodies herein may be Remicade® (Infliximab, Centocor), or Enbrel® (Etanercept®), Wyeth-Ayerst). The invention also includes bispecific antibodies that target these diseases. For example, a bispecific antibody includes anti-TNF specificity combined with the NGF-binding power of the antibody herein.

  Suitable dosages for any of the above co-administered drugs are those currently used and may be low due to the combined action (synergistic effect) of the drug and anti-NGF antibody.

  For the prevention and treatment of disease, the appropriate dosage of anti-NGF antibody is the anti-NGF antibody used, the type of disease being treated, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes. Whether it depends on medication history, patient clinical history and antibody response, and the discretion of the attending physician. Typically, the clinician will administer anti-NGF antibody until administration has achieved the desired result.

  The anti-NGF antibody is suitably administered to the patient at one time or through a series of treatments. Depending on the type and severity of the disease, for example, by one or more separate doses, or by continuous or repeated doses, the initial candidate dose for administration to a patient is about 2 mg / kg of antibody. Typical daily dosages will range from about 1 g / kg to 100 mg / kg or more, depending on the above factors. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. A preferred dosing regimen consists of administration of an initial dose of about 2 mg / kg followed by weekly maintenance administration of about 1 mg / kg anti-NGF antibody. However, other dosing regimens may be useful depending on the pattern of pharmacokinetic disruption that the practitioner wishes to achieve. The progress of this therapy is easily monitored by conventional techniques and assays.

E. Articles of Manufacture In another embodiment of the invention, an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above is provided. This product comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes and the like. The container may be formed from a material such as glass or plastic. The container contains a composition effective to diagnose and treat the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). May be). At least one active agent in the composition is an anti-NGF antibody. The container may further comprise a second pharmaceutically active agent. Preferred second agents are suitable for the treatment of inflammatory diseases such as asthma, multiple sclerosis, arthritis, lupus erythematosus and psoriasis.

  The label or package insert indicates that the composition is used to treat optimal conditions such as inflammatory conditions. In one embodiment, the label or package insert uses a composition comprising an antibody that binds NGF to show an inflammatory condition selected from the group consisting of asthma, multiple sclerosis, arthritis, lupus erythematosus, psoriasis, and the like. It shows that it can be treated. In addition, the label and package insert indicate that the patient being treated has asthma, psoriasis, arthritis or other disease or condition. Further, the article of manufacture comprises (a) an initial container containing a composition comprising an initial antibody that binds NGF and inhibits biological activity; and (b) a second that binds NGF receptor and blocks ligand activation. A second container having a composition containing a secondary antibody may be included. The article of manufacture of this embodiment of the invention may further comprise a package insert indicating that the first and second compositions can be used to treat asthma, psoriasis, arthritis or other diseases or conditions. Good. Alternatively or additionally, the article of manufacture includes a second (or third) containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution or dextrose solution. )) May be further included. It further includes other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Further details of the invention are illustrated in the following non-limiting examples.
(Example)

  Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of these cells, identified by ATCC deposit number through the examples below and throughout the specification, is the American Type Culture Collection, Manassas, Virginia.

Production and Characterization of Anti-NGF Monoclonal Antibodies A. Production of anti-NGF monoclonal antibodies This example illustrates the preparation of monoclonal antibodies that specifically bind to human NGF (hNGF). Techniques for producing monoclonal antibodies are known in the art and are described, for example, in Kohler and Milstein, Nature 256: 495-497 (1975). The experiments described in Examples 1 and 2 are further described in Hongo et al., Hybridoma 19: 215-227.

  A panel of 23 murine monoclonal antibodies against hNGF was developed by a method similar to that described by Hongo et al., Hybridoma 14: 253-260. In summary, Balbc / mouse (Charles River Laboratories, Wilmington, Delaware) was hyperimmunized with human NGF in Ribi adjuvant (Ribi Immunochem Research, Inc., Hamilton, MO). Spleen cells from mice showing the highest titer against immobilized NGF were fused with mouse melanoma cells (X63.Ag8.653; American Type Culture Collection, Rockville, Maryland). After 10-14 days, supernatants were collected and screened for antibody production by enzyme-linked immunosorbent assay (ELISA). The clone with the highest immunoreactivity after the second round of subcloning was injected into mice primed with pristane for in vivo production of MAbs (Hoogenraad et al., J. Immunol. Methods 6: 317-320 (1983) ). The ascites is pooled and affinities with staphylococcal protein A (Pharmacia) using established techniques (Moks et al., Eur. J. Biochem. 85: 1205-1210 (1986)). Purification by chromatography (Pharmacia Fast Protein Liquid Chromatography (FPLC); Uppsala, Sweden). The purified antibody preparation was aseptically filtered and stored in phosphate buffered saline (PBS) at 4 ° C.

B. Epitope Mapping Using Domain Swap Mutants Chimeric NGF / Neurotrophin-3 (NT-3) protein produced by homolog-scanning mutagenesis for epitope specificity of anti-NGF MAbs Was first determined by evaluating the binding of MAb to. The use of such domain-swap variants has clear advantages over deletion variants. Replacement of a domain with a corresponding domain that is the same size and substantially the same amino acid sequence of a related protein that is a domain-swap variant preserves secondary structure, while domain deletion preserves The following structure may be destroyed.

  Eight hNGF / hNT-3 chimeric variants (FIG. 1) with 3-7 residue substitutions of human NT-3 (hNT-3) sequence to the corresponding variable region of hNGF were oligonucleotide-directed mutagenesis Made by. This chimeric variant is transiently expressed in human 293 cells, using the purified MAb as a capture antibody and the HRP-conjugated affinity purified rabbit anti-hNGF polyclonal antibody for detection as described below Anti-hNGF binding to mutant NGF was assessed by enzyme-linked immunosorbent assay (ELISA). Binding of anti-hNGF MAb to each NGF variant was measured in 2-4 independent quantitative ELISAs and compared to binding to wild type NGF.

  In summary, microtiter plates (Nunc Maxisorb, VWR Scientific, San Francisco, Calif.) Were coated overnight at 4 ° C. with 100 L of 1 g / ml goat anti-mouse IgG (Boehringer Mannheim, Indianapolis, Indiana) per well. Excessive binding sites were blocked with PBS containing 0.05% Tween 20 and 0.5% bovine serum albumin (BSA, Intergen, San Diego, Calif .; PBS / BSA / T20). MAbs (diluted to 1 g / ml with PBS / BSA / T20) were added to appropriate wells and incubated for 1-2 hours at ambient temperature. The plate was washed and 100 L of wild type or mutant hNGF (diluted to 7.8 ng / mL in PBS / BSA / T20 to 7.8 ng / ml) was added, incubated at ambient temperature for 1-2 hours and washed again. Purified rabbit anti-hNGF polyclonal antibody conjugated with horseradish paroxidase (HRP; 1: 10,000 in PBS / BSA / T20) was added (100 L / well) and incubated for 1 hour. The plate was developed and read at a dual wavelength. The binding of MAbs to hNGF mutants was compared to wild type hNGF binding (set to 100%) analyzed under the same conditions.

High affinity for hNGF (EC ₅₀ = 0.18 nM for MAb908, 0.18 nM for MAb909, 0.37 nM for MAb911, 0.18 for MAb912, 0.74 for MAb938 and MAb 14.14 vs. 0.59) and six human anti-NGF monoclonal antibodies (MAbs 908, 909, 911, showing greater than 60-90% reduction in binding to various chimeric variants (FIG. 1) 912, 938 and 14.14) were selected for further analysis. As shown in FIG. 1, the three MAbs (908, 909 and 14.14) showed sharp region binding characteristics characterized by 60-95% loss of binding with a single variable region ( FIG. 1). Less dramatic effects of variable region variants were observed with MAb911, MAb912 and 938, which had multiple NGF variable regions contributing to the binding epitopes of these antibodies. However, the variable regions containing these epitopes are close in tertiary structure (FIG. 3A).

C. Epitope mapping using site-directed mutagenesis Previously reported to be responsible for TrkA and p75 binding and biological functions to further elucidate the epitope specificity of each of the six selected anti-NGF MAbs With particular focus on residues within the region, NGF variants exhibiting single, double, or triple amino acid point mutations were expressed and characterized (Shih et al., J. Biol. Chem., 269 (44) : 27679-27686 (1994)). The effect of the mutation on anti-NGF MAb binding was characterized by ELISA as described above. Mean EC ₅₀ values for anti-NGF MAb binding to each mutant were calculated from the ELISA binding curve and compared to the EC ₅₀ values obtained for wild type NGF binding (FIGS. 2A-F).

  With all six MAbs, the binding specificity of hNGF point mutations is generally associated with a region previously identified by loss of binding to a particular NGF chimeric mutant. However, the effect of NGF point mutations on binding of MAbs 911 and 938 is less important compared to the effects observed with other MAbs (FIGS. 2A-F). The region specificity of the mutation effect is associated with residues in or near the variable region exchanged with the NGF chimeric mutant, which mutant loses greater than 50-60% of the maximum binding of MAb 911 or 938. ing. For MAb 911, these mutants include K32A + K34A + E35A, Y79A + T81K, H84A + K88A, and R103A. Further loss of binding was observed with nearby mutants E11A, Y52A and L112A + S113A (FIG. 2C). This indicates that the epitope of MAb911 spans 4 out of 7 hNGF variable regions (FIG. 3C).

D. Epitope structure dependence on MAb binding To determine whether anti-NGF MAb binding to hNGF depends on the structural conformation of the epitope of hNGF, anti-NGF to non-reduced and reduced forms of hNGF Binding was evaluated. HNGF, either untreated or reduced with mercaptoethanol, was subjected to gel electrophoresis and transferred to a nitrocellulose blot for immunoblotting. For detection of hNGF, nitrocellulose blots were washed, incubated with primary and secondary antibodies, stirred and exposed to luminol substrate (Amersham International, Amersham, UK) for 1 hour at ambient temperature for 10-45 seconds. Exposure to x-ray film (Eastman Kodak, Rochester, New York). In FIG. 4, it can be seen that several monoclonal anti-NGF antibodies show minimal binding to the reduced form of hNGF. These include MAbs 938, 908, 911 and 912, indicating that the epitope on hNGF to which they bind is structurally affected by charge modification and reduction.

E. Molecular Modeling of Anti-NGF Monoclonal Antibody Epitopes Molecular modeling representations of identified anti-NGF MAb epitopes (FIGS. 3A-F) using the program MidasPlus (University of California San Francisco, San Francisco, California) And based on the coordinates of the tertiary structure of mouse NGF as previously described (McDonald et al., Nature, 354: 411-414 (1991)).
A summary of the MAb epitope mapping results is shown in FIG.

  The observation that several epitopes have been mapped to regions previously shown to be important in the interaction between TrkA and / or p75 and NGF will also block one or both of these interactions. Suggested deafness.

A. ¹²⁵ I-hNGF binding assay To assess the likelihood that one or more anti-NGF MAbs will block the binding of NGF to TrkA and / or p75, ¹²⁵ I-hNGF binding immunoadhesin and ¹²⁵ I The binding of -hNGF was measured in the presence of anti-NGF monoclonal antibody.

In summary, ¹²⁵ I-hNGF was prepared using a modified version of the soluble lactopaloxidase method originally described by Marchlonis (Marchlonis, Biochem J., 113: 299-305 (1969)). The final reaction mixture was fractionated on a pD-10 Sephadex G-25 size exclusion column (Pharmacia, Uprasa, Sweden) and stored at 4 ° C. Microtiter plates (Nunc, Maxisorb) were coated overnight with purified rabbit anti-human IgG-Fc specific polyclonal antibody (diluted to 2 g / ml with carbonate buffer) at 4 ° C., washed with PBS, 150 L PBS Blocked with /0.5% BSA (PBS / BSA). PBS / BSA containing human TrkA-IgG or p75-IgG immunoadhesin (kindly provided by Robert Pitti) (20 ng / mL) was added (100 L) and incubated for 1 hour at ambient temperature. HNGF diluted in PBS / BSA (150 pM final) was then added (100 L) and incubated for 1 hour at ambient temperature. Anti-NGF monoclonal antibody (667 nM 0.58 nM) or an unrelated monoclonal antibody not directed against NGF and hNGF pre-incubated overnight at 4 ° C. (150 pM final) were added simultaneously and incubated as described. Plates were washed with PBS containing 0.05% T20 and individual wells were counted in a gamma counter (Packard Cobra Model 5010, Downers, IL) for 1 minute.

As seen in FIG. 6, the anti-NGF monoclonal antibody inhibited hNGF binding to the TrkA-IgG receptor immunoadhesin, as measured by the level of ¹²⁵ I-hNGF bound to TrkA-IgG. All MAbs were assayed at the highest concentration (667 nM) and showed blocking ability, but showed different blocking ability at lower concentrations. MAbs 911, 912 and 938 showed the same blocking power (IC ₅₀ -0.5-2 nM), with 80-90% inhibition seen at 10 nM MAb or higher concentrations.

The ability of MAbs to inhibit NGF binding to the low affinity p75 receptor was assessed using p75-IgG in the binding assay described above. As shown in FIG. 7, anti-hNGF MAb uses ¹²⁵ I-hNGF to inhibit the binding of p75-IgG receptor immunoadhesin and hNGF. MAbs 911, 912 and 938 show the highest inhibitory activity with a 75-90% reduction in binding observed in the presence of MAbs lower than 1 nM. MAb 909 also showed strong blocking ability (> 90% inhibition with 10 nM MAb), whereas MAbs 908 and 14.14 were significantly less potent (FIG. 7).

B. Kinase-induced receptor activation (KIRA) assay Using the kinase-induced receptor activation (KIRA) assay, NGF-dependent TrkA self of transfected cells in response to stimulation by ligands such as hNGF and / or agonist monoclonal antibodies Phosphorylation was measured (Sadick et al., Exp. Cell Res. 234: 354-361 (1997)). Anti-NGF MAbs were evaluated for binding of TNGA extracellular domain expressed on CHO transfected cells to hNGF and subsequent ability to inhibit phosphorylation of tyrosine residues on TrkA (Figure 8). Phosphorylation of tyrosine residues was measured by ELISA using anti-phosphotyrosine MAb.

TrkA receptor with glycoprotein D fragment (gD) of herpes simplex virus, a 26 amino acid polypeptide that functions as an epitope tag from a microtiter plate (Coaster, Cambridge, Massachusetts) 1 × 10 ⁵ Chinese hamster ovary (CHO) cells expressing the extracellular domain of. Samples of either hNGF alone (150 pM) (as positive control) or hNGF (150 pM final) preincubated overnight at 4 ° C. with individual anti-NGF MAbs (667 nM 0.31 nM final) were added to TrkA expressing CHO cells (wells). Per well) and incubated at 37 ° C. for 25 minutes. An unrelated monoclonal antibody that was not directed against NGF was used as a negative control. The hNGF stimulated cells were then treated with a solubilization buffer and the lysate was treated in the same manner as described by Sadick et al. (Sadick et al., Anal. Biochem., 235: 207-214 (1996)). Treated with gD-MAb-coated microtiter plates for ELISA detection of phosphotyrosine containing. 100 l biotinylated 4G10 (Upstate Biologicals, Inc.) diluted to 0.2 mg / ml with dilution buffer (PBS containing 0.5% BSA, 0.05% Tween 20, 5 mM EDTA, and 0.01% thimerosal) (UBI Lake Placid, New York)) was added to each well. After 2 hours incubation at room temperature, the plates were washed and 100 l HRP-conjugated streptavidin (Zymed Laboratories, S. San Francisco, Calif.) Diluted 1: 50000 in dilution buffer was added to each well. The plate was agitated and incubated for 30 minutes at room temperature. The free avidin conjugate was washed away and 100 l of freshly prepared substrate solution (tetramethylbenzidine, TMB, binary substrate kit, Kirkegard and Perry, Gaitehersbug, MD) was added to each well. The reaction was continued for 10 minutes and the color development was stopped by the addition of 100 l / well of 1.0 MH ₃ PO ₄ . Absorption at 450 nm was measured using a Vmax plate reader (Molecular Devices, Palo Alto, CA) controlled by Macintosh Centris 650 (Apple Computers, Cupertino, CA) and DelataSoft sofware (BioMetallics, Inc., Princeton, NJ). _{Reading was performed at} a reference wavelength of 650 nm (A _450/650 ).

  As shown in FIG. 8, all selected anti-NGF monoclonal antibodies both bind TrkA extracellular domain expressed on CHO-transfected cells to hNGF and phosphorylate TrkA receptor tyrosine residues. Was able to be inhibited. Preincubation of MAbs 911, 912 and 938 with hNGF at the lowest concentration tested (0.3 nM) caused a maximum inhibition of approximately 60-80% of tyrosine phosphorylation relative to the control MAb. MAbs 908, 909 and 14.14 are not strong.

C. Fetal rat dorsal root ganglion survival bioassay
Another assay used to measure the effect of anti-NGF monoclonal antibodies on NGF-dependent processes was the fetal rat E14 dorsal root ganglion (DRG) survival bioassay. Anti-NGF monoclonal antibodies were evaluated for their ability to inhibit the survival effect of hNGF on fetal rat E14 dorsal root ganglion (DRG) neurons.

  Dorsal root ganglion (DRG) neurons obtained from E15 rats (6 to 8 fetuses) with additives (McMahon et al., Nat. Med. 8: 774-780 (1995)) and anti-NGF monoclonal antibody Incubated in F12 medium containing 3 ng / mL hNGF without or with it. After 72 hours incubation at 37 ° C., cells were fixed with formaldehyde and viable neurons were counted.

  FIG. 9 shows inhibition of DRG neuron survival by anti-NGF monoclonal antibodies. MAbs 908, 909 and 14.14 decreased survival only by 20-30% at the highest concentration (67 nM; 10 g / ml), whereas MAbs 911 and 912 were approximately 30-80 times lower concentrations (0.8-2.4 nM). 0.12-0.37 g / ml) inhibited survival greater than 90%. MAb 938 was able to inhibit survival activity by 90%, but was 10-30 fold less effective than MAbs 911 and 912.

  MAb911 was the most powerful blocker of NGF / TrkA interaction. MAb 911 recognizes an epitope with overlapping NGF-TrkA and p75 binding region turn A′-A ″ (V-1), and the dominant TrkA binding region of the sheet (FIG. 3D). MAb912, the second most potent NGF / TrkA inhibitor, recognizes K32, K34 and K35, a region previously shown to be important for NGF / p75 interactions. MAb 938, a more potent blocker of TrkA and p75 binding, also recognizes the N- and C-termini, a region important for TrkA or p75 binding.

  Since two of the three strongest blocking antibodies (911 and 938) have a lower affinity for NGF than weakly blocked MAbs, the difference seen in antibody blocking specificity is the MAb to hNGF It is not due to the relative affinity of.

  The NGF region important for binding to MAbs 911 and 912 overlaps with the region identified as the region important for TrkA and p75 binding. MAbs 911 and 912 can be blockers of NGF-induced activity, indicating that they are specific antagonists of in vivo activity such as inflammatory hyperalgesia.

Effect of anti-NGF on immune response On day 0 mice were immunized subcutaneously with 10 g ovalbumin and recovered. Forty days after immunization, animals were injected intraperitoneally with 10 mg / kg anti-NGF antibody 911 or control, isotype matched antibody. Two days later, the animals were boosted with ovalbumin. Immune responses were measured by ELISA in serum samples collected on day 47. As can be seen in FIG. 10, there was no significant (p> 0.05) difference in immune response between animals receiving anti-NGF antibody 911 and animals receiving control antibody. However, there was no significant trend of increased immune response in animals treated with anti-NGF monoclonal antibodies.

  In contrast, FIG. 11 shows that the following immunization with chicken gamma-globulin, treatment with anti-NGF, does not produce a significant tendency to decrease the immune response. On day 0, animals were immunized with 10 g chicken gamma-globulin and on day 40 treated with anti-NGF antibody 911 or control, isotype matched antibody (10 mg / kg IP). On day 42, animals were boosted with gamma-globulin and on day 47 serum samples were collected and analyzed by ELISA. There was no significant (p> 0.05) difference in immune response between the two groups. However, after treatment with anti-NGF monoclonal antibody 911, there was a tendency for a reduced immune response.

Effect of anti-NGF monoclonal antibody on hyperplasia
The effect of anti-NGF antibody on NGF-induced thermal hyperalgesia was investigated. In summary, adult Fischer female rats were trained 2 sessions a day by the Hargreaves test for at least 2 days prior to treatment. After habituation to the instrument and protocol, animals were randomly assigned to control or experimental groups. Both groups were tested for baseline responsiveness and then given an intraplantar injection of a 50 l volume of one foot under isoflurane anesthesia. Injections in each group contained 1.25% carrageenan in combination with either 90 g anti-NGF antibody or an inactive isotype matched control antibody.
The time for the heat to drop was measured by the Hargreaves test at 0, 2, 4, 6 and 24 hours after injection. As shown in FIG. 12, after 4 and 6 hours of treatment, thermal hyperalgesia after carrageenan injection was significantly reduced in animals treated with anti-NGF monoclonal antibody 911 compared to control animals.

Effect of anti-NGF monoclonal antibody on response to allergen Male C57 / BL6 mice (Jackson Laboratories) were treated with monoclonal anti-NGF antibody 911 (n = 16) or an isotype matched anti-gD control antibody (clone 1766; n = 16). On day 1, animals were treated with 20 mg / kg antibody, and on days 6, 13, 20, and 22, animals were treated with 10 mg / kg antibody. All antibody treatments were subcutaneous injections into the neck neck.

On days 0 and 14, half of each group of mice was 30 AU dust mite antigen (DMA; diluted with Dulbecco's PBS, then 1: 2 with ALUM, final concentration of 300 AU / ml) Sensitized (SN) by intraperitoneal injection. Non-sensitized mice (NS) received an equal volume of Dulbecco's PBS diluted 1: 2 with ALUM as a control.
Mice were stimulated with inhaled dust mite antigen (DMA) on days 21 and 22. Dulbecco's PBS supplemented with 0.01% Tween 20 for aerosolization was used to dilute house dust mites to 6000 AU / ml. All inhalation stimulants were administered in a Plexiglas pie exposure chamber. DMA was aerosolized using a PARI IS-2 nebulizer driven by 22 PSI. The nebulizer was filled with 3 ml and completed (30 minutes). The total deposited dose / exposure to the lung was ˜6.5 AU DMA.

  Mice were assayed for serum titers against dust mites along with airway hypersensitivity, cell infiltration into bronchoalveolar lavage (BAL) fluid, BAL cytokine levels, and serum IgE levels. In summary, mice were anesthetized 24 hours and 48 hours after the last stimulation, and a tracheotomy was performed with a catheter in the jugular vein. The mesh was then paralyzed with 0.28 mg / kg pancuronium and loaded onto a Plexiglas flow plethysmograph for measurement of chest expansion and airway pressure. Mice were aired using 100% oxygen at a frequency of 170 bpm and Vt equal to 9 l / g. Respiratory mechanisms (lung tolerance and dynamic compliance) were continuously monitored using the Buxco XA data acquisition program. Give mice a volume history (5 breaths at 2.5x Vt), stabilize for 2 minutes prior to baseline measurement, then use Harvard syringe pump and syringe application software The dose agonist was given 5 seconds at a time with a flow rate adjusted by weight.

Blood (serum), BAL and lungs were collected. Serum was assayed for total and specific IgE and IgG. BAL was obtained by three injections of the same aliquot of NaCl, and the BAL was assayed for total IgE by ELISA. Total white blood cells and cell differentials were obtained from BAL cells.
Treatment with anti-NGF monoclonal antibody resulted in a significant reduction in airway hypersensitivity (FIG. 13) with inflammation as measured by cell infiltration into BAL (FIG. 14). However, there is still a very high proportion of eosinophils in the BAL of anti-NGF treated animals (FIG. 15). Anti-NGF antibody treatment also reduced the level of BAL Th2 cytokine IL-13 (FIG. 16).
Despite the ability to reduce the inflammatory response to allergens, anti-NGF antibody treatment was not detected in either dust mites, either as measured by total serum immunoglobulin titers against dust mites (FIG. 17), or by serum levels of IgE (FIG. 18). Did not reduce the humoral immune response to. This suggests that the antibody did not affect the survival or function of B lymphocytes that blocked the biological effects of NGF.

  To confirm that anti-NGF antibody 911 has a functionally significant effect on NGF, 0.1, 1 or 10 mg / kg anti-NGF monoclonal antibody 911 on days 1 and 8 Or on days 1, 3, 6 and 8 at 0.1, 1 or 10 mg / kg of NGF, mice were injected subcutaneously into the neck neck. The animals were sacrificed on day 9 and the trigeminal ganglion neuropeptide CGRP levels were examined. Treatment with 1 or 10 mg / kg of NGF resulted in an increase in CGRP, confirming that this peptide was controlled by NGF levels (FIG. 19). Treatment with 1 or 10 mg / kg of anti-NGF monoclonal antibody 911 resulted in a decrease in ganglion CGRP content (FIG. 19), and this dose resulted in functional endogenous NGF when an immune response occurred in the above experiment. It was confirmed that significant blockage occurred.

6 outlines the ability of six anti-NGFs to bind to NGF / NT-3 chimeric variants. The relative binding of each MAb to the NGF / NT3 mutant has been compared to the binding of wild-type hNGF: (−), <10%; (+), 10-30%; (++), 30− 60%; (+++), 60-100%. The EC ₅₀ of each MAb for binding to hNGF is: MAb908, 1.8x10 ^-10 M; MAb911, 3.7x10 ^-10 M; MAb912, 1.8x10 ^-10 M; MAb938, 7.4x10 ^-10 M; MAb14.14, 5.9x10 ^-10 M. Shows binding of MAb to wild type and mutant hNGF. The average EC ₅₀ value of each mutant obtained in 2 to 5 independent ELISAs was compared to the EC ₅₀ value obtained for wild type NGF binding. EC ₅₀ values were determined by linear regression analysis using the Kaleidagraph software program (Abelbeck Software). Mutants that caused at least a 2-fold reduction in MAb binding were indicated by striped bars and the contributing residues were labeled. Shows binding of MAb908 to wild type and mutant NGF. Shows binding of MAb909 to wild type and mutant NGF. Shows binding of MAb911 to wild type and mutant NGF. Shows binding of MAb912 to wild type and mutant NGF. Shows binding of MAb 938 to wild type and mutant NGF. Shows binding of MAb14.14 to wild type and mutant NGF. Molecular models of MAb epitopes on NGF of anti-NGF MAbs 908, 909, 911, 912, 938 and 14.14, respectively, are shown. NGF monomers are distinguished by light and medium gray display, and residues identified by ELISA that act on MAb binding are shown in black. In FIG. 3A, each variable region is labeled. Figure 2 shows an immunoblot analysis of anti-NGF binding to non-reduced hNGF (A) and reduced hNGF (B). The molecular weight marker can be visually confirmed in the first lane. Buffer and negative control antibodies are shown in lanes 2 and 3, respectively. Under non-reducing conditions, purified hNGF migrates as three sets of bands, which are consistent with monomer, dimer and partially processed dimer hNGF. 6 summarizes the results of MAb epitope mapping. It has been shown that anti-NGF MAbs inhibit hNGF binding to TrkA-IgG receptor immunoadhesins. FIG. 6 shows the ability of anti-NGF MAbs to inhibit hNGF binding to p75-IgG immunoadhesins. FIG. 6 shows the ability of anti-NGF MAbs to inhibit hNGF binding to TrkA extracellular domain expressed in transfected CHO cells. Inhibition of tyrosine phosphorylation was measured by ELISA using anti-phosphotyrosine MAb. Figure 2 shows the ability of anti-NGF MAbs to inhibit the survival effect of hNGF on embryonic rat dorsal root ganglion neurons. Maximum viability was based on the signal obtained with NGF alone and the highest inhibition determined by the signal obtained with the addition of a saturating concentration of soluble TrkA-IgG. It shows that treatment with anti-NGF MAb911 did not significantly enhance the immune response to ovalbumin. It shows that after immunization with chicken gamma-globulin, treatment with anti-NGF MAb 911 did not significantly reduce the immune response. It shows that treatment with anti-NGF MAb 911 blocks NGF-induced thermal hyperalgesia. In sensitized C57 / BL6 mice (SN), treatment with anti-NGF MAb911 is shown to inhibit airway hypersensitivity after stimulation with inhaled dust mite antigen. In sensitized C57 / BL6 mice (SN), treatment with anti-NGF MAb reduces leukocyte, lymphocyte and eosinophil infiltration into BAL following stimulation with inhaled dust mite antigen . In anti-NGF MAb-treated sensitized mice, BAL cell infiltration is reduced, but the percentage of eosinophils remains high. It shows that treatment with anti-NGF MAb911 stops the increase of IL-13 in BAL after antigen stimulation. Despite its ability to reduce the inflammatory response to allergens, treatment with anti-NGF MAbs does not reduce the humoral immune response as measured by total serum immunoglobulin titers against house dust mites. It shows that treatment with anti-NGF MAb also does not reduce the total serum level of IgE. We show that treatment with anti-NGF MAb increases the level of CGRP in the trigeminal ganglion while treatment with NGF causes a decrease in CGRP levels.

Claims (47)

Administering to a human patient an effective amount of an anti-human NGF (anti-hNGF) monoclonal antibody that has no significant side effects on the patient's immune system and can bind to hNGF with an affinity in the nanomolar range; A method of controlling NGF-related disease in a human patient comprising inhibiting the binding of human TrkA (hTrkA) and hNGF in vivo. The method of claim 1, wherein the binding affinity of the antibody to hNGF is from about 0.10 to about 0.80 nM. The method of claim 2, wherein the binding affinity of the antibody to hNGF is from about 0.15 to about 0.75 nM. 4. The method of claim 2, wherein the binding affinity of the antibody to hNGF is from about 0.18 to about 0.72 nM. 2. The method of claim 1, wherein the antibody binds essentially the same hNGF epitope as an antibody selected from the group consisting of MAb911, MAb912 and MAb938. 6. The method of claim 5, wherein said antibody binds essentially the same hNGF epitope as antibody MAb911. 2. The method of claim 1, wherein the antibody can also bind to mouse NGF (muNGF). The method of claim 1, wherein the antibody is an antibody fragment. The antibody fragment is selected from the group consisting of multispecific antibodies formed from Fab, Fab ′, F (ab ′) ₂ , Fv fragments, diabodies, single chain antibody molecules and antibody fragments. 8 methods. 10. The method of claim 9, wherein the single chain antibody molecule is a single chain Fv (scFv) molecule. The method of claim 1, wherein the antibody is chimeric. 2. The method of claim 1, wherein the antibody is humanized. The method of claim 1, wherein the antibody is a human antibody. The method of claim 1, wherein the antibody is a bispecific antibody. 15. The method of claim 14, wherein the bispecific antibody is anti-IgE specific. 2. The method of claim 1, wherein the NGF-related disease is other than a disease associated with the action of NGF on the nervous system. The method of claim 16, wherein the NGF-related disease is an inflammatory condition. 18. The method of claim 17, wherein the inflammatory condition is selected from the group consisting of asthma, multiple sclerosis, arthritis, lupus erythematosus and psoriasis. 19. The method of claim 18, wherein the symptom is asthma. 19. The method of claim 18, wherein the symptom is arthritis. 21. The method of claim 20, wherein the arthritis is rheumatoid arthritis. 19. The method of claim 18, wherein the symptom is psoriasis. 18. The method of claim 17, wherein the antibody is administered in combination with other therapeutic agents for the treatment of the inflammatory condition. 20. The method of claim 19, wherein the antibody is administered in combination with a corticosteroid. 25. The method of claim 24, wherein the corticosteroid is beclomethasone dipropionate (BDP). 20. The method of claim 19, wherein the antibody is administered in combination with an anti-IgE antibody. 27. The method of claim 26, wherein the anti-IgE antibody is rhuMAb-E25 or rhuMAb-E26. 24. The method of claim 21, wherein the antibody is administered in combination with other therapeutic agents for the treatment of rheumatoid arthritis. 29. The method of claim 28, wherein the therapeutic agent is an anti-TNF antibody or antibody or immunoadhesin that specifically binds to a TNF receptor. Binding of hNGF to human TrkA (hTrkA) in vivo comprising a chimeric, humanized or human anti-human NGF (anti-hNGF) monoclonal antibody capable of binding hNGF with an affinity in the nanomolar range Wherein the antibody has no significant side effects on the patient's immune system in combination with a pharmaceutically acceptable carrier. 32. The pharmaceutical composition of claim 30, wherein the antibody is an antibody fragment. The antibody fragment is selected from the group consisting of multispecific antibodies formed from Fab, Fab ′, F (ab ′) ₂ , Fv fragments, diabodies, single chain antibody molecules and antibody fragments. 31 pharmaceutical compositions. 32. The pharmaceutical composition of claim 30, wherein the antibody is a bispecific antibody. 34. The pharmaceutical composition of claim 33, wherein said bispecific antibody is capable of specifically binding to native human IgE. 34. The pharmaceutical composition of claim 33, wherein said bispecific antibody is capable of specifically binding to natural human TNF or a natural human TNF receptor. 32. The pharmaceutical composition of claim 30, further comprising other pharmaceutically active ingredients. 37. The pharmaceutical composition of claim 36, wherein said other pharmaceutically active ingredient is suitable for treating inflammatory conditions. 38. The pharmaceutical composition of claim 37, wherein the inflammatory condition is selected from the group consisting of asthma, multiple sclerosis, arthritis, lupus erythematosus and psoriasis. 40. The pharmaceutical composition of claim 38, wherein the inflammatory condition is asthma. 40. The pharmaceutical composition of claim 38, wherein the inflammatory condition is arthritis. 41. The pharmaceutical composition of claim 40, wherein the arthritis is rheumatoid arthritis. 42. The pharmaceutical composition of claim 41, wherein the inflammatory condition is psoriasis. container;
32. A product comprising: the pharmaceutical composition of claim 30; and instructions for using the composition for controlling NGF-related disease in a human patient. 44. The article of manufacture of claim 43, further comprising a second pharmaceutically active ingredient. 45. The article of manufacture of claim 44, wherein said second pharmaceutically active ingredient is suitable for the treatment of inflammatory conditions. 46. The article of manufacture of claim 45, wherein the inflammatory condition is selected from the group consisting of asthma, multiple sclerosis, arthritis, lupus erythematosus and psoriasis. 44. The article of manufacture of claim 43, wherein the composition further comprises a second container that comprises a secondary antibody that binds to the NGF receptor and blocks ligand activation, and wherein the composition is contained.

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